Mapping discontinuous epitopes recognized by human enhancing antibodies In the last decade, mass spectrometry has been employed by more and more researchers for identifying the proteins in a macromolecular complex as well as for defining the surfaces of their binding interfaces. This characterization of protein:protein interfaces usually involves at least one of several different methodologies in addition to the actual mass spectrometry. For example, limited proteolysis is often used as a first step in defining regions of a protein that are protected from proteolysis when the protein of interest is part of a macromolecular complex. Other techniques used in conjunction with mass spectrometry for determining regions of a protein involved in proteinprotein interactions include chemical modification, such as covalent cross-linking, acetylation of lysines, hydrogen-deuterium exchange, or other forms of modification. We have been working on identifying the antigen recognized by two neutralizing antibodies: 1) MAb 559/64-D;and 2) MAb . MAb 559/64-D is a neutralizing anti-HIV antibody that recognizes a discontinuous epitope on the envelope protein gp120 of the human immunodeficiency virus that is exposed during conformational changes when gp120 binds to its primary receptor CD4. Based on chemical modification of lysine and arginine residues followed by mass spectrometric analysis, we determined the epitope on gp120 recognized by the human monoclonal antibody 559/64-D which was previously found to be specific for the CD4 binding domain. Twenty-four lysine and arginine residues in recombinant full-length glycosylated gp120 were characterized;the relative reactivities of two lysine residues and five arginine residues were affected by the binding of 559/64-D. The data show that the epitope is discontinuous and is located in the proximity of the CD4-binding site. Additionally, the reactivities of a residue that is located in the secondary receptor binding region and several residues distant from the CD4 binding site were also altered by Ab binding. These data suggest that binding of 559/64-D induced conformational changes which result in altered surface exposure of specific amino acids distant from the CD4-binding site.. Consequently, binding of 559/64-D to gp120 affects not only the CD4-binding site, which is recognized as the epitope, but appears to have a global effect on surface exposed residues of the full-length glycosylated gp120. MAb 2909 recognizes a discontinuous epitope on the native trimeric spike conformation of the HIV envelope protein. It does not recognize monomeric gp120 or gp160. The trimeric structure of the functional envelope protein raises a significant problem for the determination of the epitope recognized by this Ab. Over-expression of gp160 leads to cleavage of the gp41 and gp120 interaction. Several research groups have described the incorporation of Cys into the variable regions of gp120 which can lead to disulfide bond formation and stabilization of the trimer. Based on this information, we are currently having a stable trimer expression system developed.